PROJECT ABSTRACT Resistance to tyrosine kinase inhibitors (TKI) in lung cancer (LC) is often connected to cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment (TME). CAFs can cause drug resistance via secretion of growth factors as well as direct contact with cancer cells. Furthermore, tumor cells educate TME fibroblasts to adapt a CAF phenotype, leading to complex and bi-directional signaling between cancer cells and CAFs. Importantly, TKIs do not simply shut down oncogenic signaling, but lead to an adaptive rewiring of the signaling network. In addition, most TKIs have multiple targets, and TKI off-targets can have important effects on efficacy and response, either by restricting or boosting it. This is not limited to cancer cells, but TKIs can simultaneously engage proteins and signaling pathways in cancer as well as stromal cells. Together, these scenarios create a highly dynamic, bi-directional and drug-specific adaptive signaling response of the cancer cell/CAF system, which results in modulation of drug sensitivity and development of drug-tolerant ?persister? cell populations. We hypothesize a) that individual TKIs elicit drug- and cell-specific adaptive signaling responses and resistance mechanisms in the LC cell/CAF system, and b) that disrupting bi-directional signaling between LC cells and CAFs can enhance drug sensitivity and eliminate CAF-supported persister cells. Using unbiased, cell type-specific proteomics approaches, we will test these hypotheses in the following specific aims: 1) To characterize mechanisms and roles of fibroblast RTK pathway activation by cancer cells. Genetically activating RTK-driven signaling pathways inside CAFs will allow the characterization of bi-directional signaling of CAFs and LC cells using ?cell type-specific labeling using amino acid precursors? (CTAP)-based phosphoproteomics and how it in turn affects LC cell proliferation, invasion and drug sensitivity. Relevant signaling pathways will be evaluated by proximity-ligation assays on patient-derived tissue microarrays (TMAs) and in orthotopic, heterotypic in vivo models. 2) To develop strategies to functionally engage TKI-induced adaptive signaling in CAFs and LC cells. Using CTAP-based chemical and phosphoproteomics, we will determine LC- and CAF-specific adaptive signaling responses and target profiles of clinical TKIs. Functional validation by RNAi and rescue experiments will identify CAF targeting drugs. Synergy with TKI will be evaluated in co-culture and in patient-derived xenograft (PDX) models. The approach is innovative, because it represents a novel way of targeting cancer by developing strategies to simultaneously engage signaling pathways in cancer cells as well as the TME, which are enabled by application of state-of-the-art proteomics. The proposed research is significant as it will transform our understanding of the dynamics and complexity of signaling evoked by LC cell-CAF interactions and the roles of these circuits in drug resistance and sensitization. Further, these studies will reveal conceptually novel opportunities for clinical development of targeted combination therapies for a patient population with a significant unmet medical need.